TDP-43 regulates cholesterol biosynthesis by inhibiting sterol regulatory element-binding protein 2

Dyslipidemia is considered an essential component of the pathological process of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease. Although TAR DNA Binding Protein 43 kDa (TDP-43) links both familial and sporadic forms of ALS and cytoplasmic aggregates are a hallmark of most cases of ALS, the molecular mechanism and the in vivo relation of ALS dyslipidemia with TDP-43 have been unclear. To analyze the dyslipidemia-related gene expression by TDP-43, we performed expression microarray and RNA deep sequencing (RNA-Seq) using cell lines expressing high levels of TDP-43 and identified 434 significantly altered genes including sterol regulatory element-binding protein 2 (SREBP2), a master regulator of cholesterol homeostasis and its downstream genes. Elevated TDP-43 impaired SREBP2 transcriptional activity, leading to inhibition of cholesterol biosynthesis. The amount of cholesterol was significantly decreased in the spinal cords of TDP-43-overexpressed ALS model mice and in the cerebrospinal fluids of ALS patients. These results suggested that TDP-43 could play an essential role in cholesterol biosynthesis in relation to ALS dyslipidemia.

Immunocytochemistry. Cells were fixed in 4% paraformaldehyde (pH 7.4) for 10 min at room temperature and rinsed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by rinsing with PBS. Nonspecific binding was blocked with Blocking One HIST (Nacalai, Kyoto, Japan) for 10 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, and then labeled with appropriate fluorescent-tagged secondary antibodies. DAPI (Thermo Fisher Scientific) was used to label nuclei. Acquisition of fluorescence images and quantification were performed using In Cell www.nature.com/scientificreports/ Analyzer 6500 (GE Healthcare). The following primary antibodies were used in this assay: SREBP2 (Abcam, Emeryville, CA, 1:50).
Equipment and settings. The image acquisition tool LAS 4000 (GE Healthcare) was used for immunoblots, and images were processed on imageJ (https:// imagej. nih. gov/ ij/) software.
Reporter assay. HEK293T  Mice. Prp-TDP43A315T mice (B6Cg-Tg (Prnp-TARDBP*A315T) 95Balo/J, 010700) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). As described previously 20 , their asymptomatic stage is between 1 and 2 months of age. The onset of gait disorder appeared at ∼ 3 months of age in transgenic mutant TDP-43 mice and increased little by little over the next few months. The animals became paralyzed at ∼ 5 months of age, corresponding to symptomatic stage (gender-independent average); they were unable to move their hindlimbs or right themselves when placed on their backs. For cholesterol measurements, at 3 months of age (pre-symptomatic stage) and 4-6 months of age (symptomatic stage) the mice were perfused with cold PBS and spinal lipids were extracted with chloroform/methanol Cholesterol measurement. For quantitative analysis of cholesterol, cells were homogenized in PBS and lipids were extracted by modified Bligh and Dyer extraction method 21,22 . Briefly, 100 μl of chloroform plus methanol (2:1) was added to 100 μl of PBS including homogenized cells after counting the cell number. After centrifugation, the chloroform layer was collected and dried with a centrifugal dryer. The dry matter was lysed in 100 μl of ethanol to measure free cholesterol using a Cholesterol Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Fluorescence signals were read with an Envision Multilabel Reader (PerkinElmer). Total lipids including sterols in spinal fluids from patients were extracted by the same method, with the extracts being subjected to LC-MS/MS-based quantification (Agilent 6400). Briefly, 100 μl of chloroform plus methanol (2:1) was added to 10 μl of spinal fluids mixed with 90 μl of PBS. After centrifugation, the chloroform layer was collected and dried with a centrifugal dryer. The dry matter was then lysed in 100 μl of ethanol, filtered with a 0.2-μm centrifugal filter tube, and measured for cholesterol by the use of Agilent 6400.

Statistics.
All data are shown as mean ± s.e.m. or ± s.d. Two group-analyses were performed using unpaired two-tailed Student's t-test or paired t-test. One-or two-way ANOVA was performed for each comparison, followed by Tukey's post hoc tests for evaluation of pair-wise group differences. A p value < 0.05 was considered statistically significant. In a comparative analysis of the amount of cholesterol, an alternative hypothesis was applied, namely that sample data could differ between the two groups by 10% (α = 0.05, β = 0.2, power = 0.8 and effect size = 10% of the average), and the sample size was then determined. Analyses were performed by JMP (SAS Institute Inc., Cary, NC, USA) and Excel Tokei (Social Survey Research Information, Tokyo, Japan).

Ethics approval and consent to participate. This study was approved by the Institutional Review
Boards of Kyoto University and Tokushima University (Approval number: 2572-6, reference year: 2022), and all surgical procedures for animal studies were performed according to the rules set forth by the Ethics Committee of Kyoto University (Approval number: 17-91-11, reference year: 2022). Written informed consent was received from the participants prior to inclusion in the study. Samples from the participants were identified by numbers, not by names.
Consent for publication. Written informed consent for publication was received from each participant.

Gene expression and transcriptome profiling under high levels of TDP-43.
To analayze dyslipidema-related gene expression by TDP-43, we performed expression microarray and RNA sequencing (RNAseq) using 293 T Rex cells stably expressing doxycycline-inductive DAP-TDP-43 (SI: Supplementary Fig. 1A) 19 . The microarray showed that elevated TDP-43 downregulated the expression of genes related to cholesterol biogenesis, including SREBP2, HMGCS1 and LDLR, with a significantly decreased cholesterol metabolic process as a www.nature.com/scientificreports/ gene ontology (GO) term ( Fig. 1A and SI: Table S3). SREBP2 was a top upstream transcriptional regulator of the altered gene expressions caused by overexpressed TDP-43 (SI : Table S4). QRT-PCR showed that TDP-43 reduced the mRNA levels of genes related to cholesterol biogenesis including HMGCS1, HMGCR and LDLR as well as SREBP2 (Fig. 1B). We further performed RNAseq and identified 434 genes that were significantly altered under doxycycline-inductive TDP-43 (Tukey's test, p < 0.05, SI: Table S5). Among them, elevated TDP-43 significantly decreased the expression levels of HMGCS1, HMGCR, DHCR7 and SREBP2 without biasing the splicing variants of SREBP2 ( Fig. 1C-E). Taken together, TDP-43 could regulate the expression of genes related to cholesterol biosynthesis and their master regulator SREBP2.
Overexpressed TDP-43 decreases cholesterol biogenesis by inhibiting SREBP2 activity. SREBP2 is a critical transcriptional factor that regulates various enzymes in the cholesterol biosynthetic pathway by binding sterol-regulatory elements (SREs) in the promoters of cholesterol-related genes 23,24 . Western blotting revealed that TDP-43 significantly and persistently decreased the amount of the cleaved N-active form of SREBP2 (N-SREBP2) in a TDP-43 overexpression condition ( Fig. 2A,B). In accordance with the suppressive effect on SREBP2 quantity, TDP-43 negatively regulated the endogenous transcriptional activity of SREBP2 as measured with a transfected SRE-luciferase construct (SRE-luc) (Fig. 2C). Elevated TDP-43 could inhibit SRE sensitivity to a decreased cholesterol level in a fashion similar to authentic sterol-regulated conditions such as cholesterol depletion by the addition of methyl-beta-cyclodextrin (MbCD) and lovastatin, HMGCoA reductase inhibitors (Fig. 2D). TDP-43 overexpression altered the amount of cholesterol (Fig. 2E). Immunocytochemistry showed that the area of nucleus-localized SREBP2 was significantly decreased compared to control when TDP-43 was overexpressed (Fig. 2F,G). It is known that SREBP2 could be modified by several post-translational mechanisms (Fig. 3A). Thus, we examined the expression levels of proteases, S1P and S2P, which cleave N-SREBP2 from the full-length SREBP2, and found that, except Insig-1, there was no remarkable difference in the two expression levels between TDP-43 abundant condition and cholesterol-depleted condition (Fig. 3B). There was also no decrement in the endoplasmic reticulum (ER) anchor protein such as SCAP protein level. The expression levels of these factors related to cholesterol biosynthesis other than N-SREBP2 and Insig-1 varied between control and TDP-43 overexpressed condition (Fig. 3C). An immunoprecipitation assay using HEK293T cells revealed that TDP-43 did not bind to N-SREBP2 (Fig. 3D). Taken together, overexpressed TDP-43 could impair cholesterol biosynthesis by inhibiting SREBP2 activity.  Table 1). The cholesterol amounts were significantly decreased in the CSF of ALS patients, but not in their serum (Fig. 5 and Tables 2, 3).

Discussion
Sterol regulation for maintaining cellular cholesterol is critical for cell growth and survival. In general, neurons may not be the main cell type biosynthesizing cholesterol in the central nervous system (CNS), as astrocytes and oligodendrocytes are the main producers and suppliers of cholesterol in the CNS 26 . TDP-43 deficiency dysregulates SREBP2 and induces demyelination in oligodendrocytes 27 , suggesting that a loss of function of TDP-43 in CNS could play an important role in ALS pathogenesis.
We found that overexpressed TDP-43 decreased the expression of genes related to cholesterol biosynthesis and impaired the transcriptional activity of SREBP2 (SI: Supplementary Fig. 1B). However, our study has several limitations, as follows.
First, the exact mechanism by which TDP-43 overexpression may regulate SREBP2 expression and functionality still remains unresolved. The N-terminal transcription domain of SREBP2 was more affected by TDP-43 overexpression, while the full-length SREBP2 was unaffected ( Fig. 2A). Thus, the apparent reduced SREBF2 mRNA could not account for the observed reduction in N-terminal SREBP2. We examined the expression levels of the factors related to cholesterol biosynthesis between control and TDP-43 overexpressed condition. We found that, except for Insig-1, they varied in several experiments (Fig. 3B,C). We speculated that they may affect each other and vary in the context of the homeostasis of cholesterol biosynthesis. We examined previous TDP-43 CLIP analysis 28 regarding whether TDP-43 could bind to the transcripts whose expression levels decreased by TDP-43 overexpression. TDP-43 binds to mRNA of SREBP2 and HMGCR but not to HMGCS1 or DHCR7. Further study is needed to elucidate how TDP-43 could alter these selected transcripts by its binding. Therefore, additional mechanisms by TDP-43 overexpression could contribute to a reduced SREBP2 mRNA level and N-terminal SREBP2 level.
Second, we found a modest yet significant difference in both total and free cholesterol in the spinal cord between control and ALS model mice, although only in female mice. A previous study revealed that male mice www.nature.com/scientificreports/ www.nature.com/scientificreports/ die of bowel obstruction due to gut dysfunction 25 , which could have masked the metabolic phenotypes in our study, and by this the cause of death. Third, we could not identify the mechanism by which cholesterol levels were decreased in ALS patient CSF but not in the serum. It is also known that there is an increase in TDP-43 levels in ALS spinal fluid, and a negative correlation between elevated TDP-43 and cholesterol has been suggested in ALS patient CSF 29 . We speculate that abundant TDP-43 in CNS could specifically decrease the cholesterol biogenesis in ALS patient CSF.
It has been reported that the level of HMG-CoA reductase is reduced in ALS spinal cord grey matter, and that the SREBP2 expression level is lowered in ALS model mice with mutant SOD1 30 , and also that Spinocerebellar Ataxia type 2 model mice harboring TDP-43 pathology exhibit concomitant cholesterol biogenesis suppression 31 . Our observations suggest that TDP-43 impaired cholesterol biosynthesis, which is presumably involved in other neurodegenerative diseases including Alzheimer's disease (AD) and Huntington's disease 16,32,33 .
We speculate that a cholesterol complement may not be enough to rescue ALS MNs. Olesoxime, which has a cholesterol-like structure, has been thought to be neuroprotective for ALS MNs 34 . However, this compound failed in a phase 3 clinical trial for ALS 35 , which could underlie the requirement for a concomitant therapy that compensates for cholesterol in ALS MNs. Furthermore, as shown in Huntington's disease, SREBP2 gene therapy may be promising for ALS 36 . Taken together, this reverse-translational study could provide a molecular basis for future ALS therapy targeting cholesterol biosynthesis.   www.nature.com/scientificreports/